The research supported by this grant will focus on structure-function relationships of flavoprotein monooxygenases using biochemical, biophysical and protein engineering methods. A major goal is to develop an understanding in molecular terms of how the different families of flavoprotein monooxygenases can all function through the formation of common oxygen intermediates, the C4a-peroxyflavins, but nevertheless have very diverse substrate specificities and mechanisms of regulation. Because the flavin provides a sensitive built-in spectroscopic reporter group for monitoring individual stages of the reactions, a very detailed examination of the individual reactions, including protein dynamic events, is possible. It is planned to study in detail four main classes of such enzymes: 1) aromatic hydroxylases, which appear to function using the flavin hydroperoxide as a nucleophile; 2) cyclohexanone monooxygenase, an enzyme incorporating an oxygen atom into its substrate by a Baeyer-Villiger reaction, using the flavin C4a peroxide anion as a nucleophile; 3) broad substrate-tolerant enzymes capable of oxygenating a diverse array of nitrogen-and sulfur-compounds; 4) unusual 2-protein component enzymes from bacterial sources that carry out the hydroxylation of p-hydroxyphenylacetate. These enzymes are widely distributed in nature and play important roles in xenobiotic metabolism in animals, biosynthesis of plant hormones and bioremediation processes by soil bacteria. They also offer the possibility of synthesis of chiral compounds that could be useful in drug development. Extensive use will be made of site-directed mutagenesis, based on structural information already available for p-hydroxybenzoate hydroxylase and phenol hydroxylase, and determination of the structure of cyclohexanone monooxygenase is also planned. The reaction mechanism of each enzyme will be explored by rapid reaction techniques, including stopped-flow fluorescence and absorbance spectroscopy, by NMR of isotopic ally labeled protein, and by solid state NMR techniques. Extensive use will also be made of artificial flavins introduced into the enzymes in place of the natural flavins, as probes both of active site structure and mechanism.